Despite strict quality control of thymocytes subjected to positive and negative selection in the thymus, a number of autoreactive T cells escape into the periphery. CD25+CD4 regulatory T cells (Treg) play a key role in prevention of autoimmunity. Recent observations of significantly increased Treg numbers in mice coexpressing a transgenic TCR and its cognate antigen suggested that these cells have increased affinity for self class II-bound peptides. However, peptide specificity of TCR expressed in naturally arising Treg, and requirements for the recognition of specific self-peptides at different stages of Treg differentiation remain a major unknown in the field. This lack of knowledge impedes further progress in our understanding of the development and function of this crucial T cell subset. Our preliminary studies suggested that a substantial proportion of Treg is specific for ubiquitously expressed class II-bound peptides present in high copy numbers. Based on these data and our recent observation of MHC class II-dependent proliferation of Treg in a lymphopenic host, we further hypothesize that Treg TCR interactions with the MHC-peptide ligands are required for the peripheral maintenance of the Treg subset and its function. In this proposal, we will test these hypotheses by characterizing Treg specificity, and by further investigating requirements for self-peptide recognition for Treg development in the thymus and for their function in the periphery. In the former experiments we will attempt a direct identification of the peptide-MHC class II complexes recognized by Treg TCR. In the latter studies, we will be using transgenic Treg TCR selected in "single peptide" mice and in mice with a wild type repertoire of class II-bound self peptides, as well as polyclonal Treg from "single peptide" mice. These studies will provide new information on the role of Treg recognition of specific MHC class II-bound peptides in Treg thymic development, maintenance in the periphery, and in Treg-mediated protection against autoimmunity. This knowledge is of principal importance for understanding of Treg biology and for developing Treg cell therapies to alleviate autoimmunity and transplant rejection.